Epidemiologic and laboratory studies implicate Vitamin D (VitD) in both reducing the risk of developing colorectal cancer (CRC) and improving survival among patients with established CRC. VitD complexes with the intracellular VitD receptor (VDR) to regulate target genes, but the mechanisms behind the significant influence on CRC development and progression are unknown. Moreover, no study has adequately examined the impact of pharmacologic VitD supplementation on outcome in patients with CRC. During the last funding period our dose-finding study of VitD supplementation in African Americans determined that 4000 lU of VitD3 daily is required to raise plasma VitD levels into the optimal range associated with cancer prevention. In this continuation project, we propose a cohesive laboratory and clinical effort using cell lines, mouse models, and human patients to define the mechanism and basis of VitD and VDR action in CRC biology, to address causality, and to assess the role of VitD supplementation on CRC patient outcomes in a randomized intervention trial. In Aim 1 we will use genetically engineered mice to test the hypothesis that VitD suppresses CRC development and progression through direct actions on intestinal mucosal cells. We will use chromatin immunoprecipitation and massively parallel sequencing (ChlP-seq) to identify VitD-VDR transcriptional targets in CRC cell lines and in human colon cancers and metastases collected in an innovative preoperative trial of VitD supplementation. We will apply innovative computational tools to identify bona fide targets and key VitD-dependent pathways. Comparisons of VDR occupancy profiles in normal and cancerous colon in the same individual, and between VitD- or placebo-treated tumors, will deliver a refined understanding of target genes relevant to CRC, allowing rapid translation of in vitro findings to the clinic. The cornerstone of Aim 2 is a prospective, randomized, double-blind phase II clinical trial to test the hypothesis that, in conjunction with standard chemotherapy, pharmacologic doses of VitD3 improve progression-free survival in patients with metastatic CRC. We will use powerful information from Aim 1 on VitD-VDR genomic binding sites and candidate target genes to examine their roles in differential VitD sensitivity within the clinical trial. Furthermore, we will determine whether polymorphisms in these binding sites and regulated genes are associated with greater or lesser response to VitD in CRC progression in humans, and assess whether supplemental VitD3 confers greater benefit when tumors overexpress VDR or carry wild-type KRAS This translational project leverages multidisciplinary expertise and complementary approaches to identify molecular mechanisms, novel biomarkers, and optimal clinical strategies to integrate supplemental VitD in CRC prevention and treatment. Our findings will meaningfully advance strategies for CRC prevention and treatment, including routine monitoring of VitD status and incorporation of VitD supplementation in patient care, resulting in rapid and measurable reductions in CRC incidence and mortality.